生物技术通讯
生物技術通訊
생물기술통신
LETTERS IN BIOTECHNOLOGY
2014年
6期
792-795
,共4页
朱晓明%李盼%王琳%王宇%张亮%徐元基%杜芝燕%于继云%阎瑾琦
硃曉明%李盼%王琳%王宇%張亮%徐元基%杜芝燕%于繼雲%閻瑾琦
주효명%리반%왕림%왕우%장량%서원기%두지연%우계운%염근기
萤光素酶%pEE14.1载体%活体成像
螢光素酶%pEE14.1載體%活體成像
형광소매%pEE14.1재체%활체성상
luciferase%pEE14.1 vector%in vivo imaging
目的：为研究10 kb以上DNA疫苗质粒的体内电穿孔递送，构建萤光素酶报告基因表达质粒pEE14.1-luc，并验证其表达。方法：从质粒pGL3-CMV中通过酶切、补平和纯化的方法获得萤光素酶基因luc，克隆入pEE14.1载体，构建重组表达质粒pEE14.1-luc，瞬时转染293T细胞，采用Western印迹、流式细胞术和免疫荧光等方法对萤光素酶基因在体外的表达情况进行验证，并运用活体成像仪检测萤光素酶基因在小鼠活体内的表达。结果：经菌液PCR鉴定和测序验证，pEE14.1-luc与预期设计完全一致；流式细胞术检测luc阳性表达率为22.41%，免疫荧光检测可见绿色荧光表达，Western印迹检测在相对分子质量为62×103处显现目的蛋白条带；同时，利用活体成像技术也检测到萤光素酶基因在小鼠活体内的表达。结论：pEE14.1-luc表达质粒构建成功，为研究DNA疫苗体内表达机制和体内电穿孔递送条件优化奠定了基础。
目的：為研究10 kb以上DNA疫苗質粒的體內電穿孔遞送，構建螢光素酶報告基因錶達質粒pEE14.1-luc，併驗證其錶達。方法：從質粒pGL3-CMV中通過酶切、補平和純化的方法穫得螢光素酶基因luc，剋隆入pEE14.1載體，構建重組錶達質粒pEE14.1-luc，瞬時轉染293T細胞，採用Western印跡、流式細胞術和免疫熒光等方法對螢光素酶基因在體外的錶達情況進行驗證，併運用活體成像儀檢測螢光素酶基因在小鼠活體內的錶達。結果：經菌液PCR鑒定和測序驗證，pEE14.1-luc與預期設計完全一緻；流式細胞術檢測luc暘性錶達率為22.41%，免疫熒光檢測可見綠色熒光錶達，Western印跡檢測在相對分子質量為62×103處顯現目的蛋白條帶；同時，利用活體成像技術也檢測到螢光素酶基因在小鼠活體內的錶達。結論：pEE14.1-luc錶達質粒構建成功，為研究DNA疫苗體內錶達機製和體內電穿孔遞送條件優化奠定瞭基礎。
목적：위연구10 kb이상DNA역묘질립적체내전천공체송，구건형광소매보고기인표체질립pEE14.1-luc，병험증기표체。방법：종질립pGL3-CMV중통과매절、보평화순화적방법획득형광소매기인luc，극륭입pEE14.1재체，구건중조표체질립pEE14.1-luc，순시전염293T세포，채용Western인적、류식세포술화면역형광등방법대형광소매기인재체외적표체정황진행험증，병운용활체성상의검측형광소매기인재소서활체내적표체。결과：경균액PCR감정화측서험증，pEE14.1-luc여예기설계완전일치；류식세포술검측luc양성표체솔위22.41%，면역형광검측가견록색형광표체，Western인적검측재상대분자질량위62×103처현현목적단백조대；동시，이용활체성상기술야검측도형광소매기인재소서활체내적표체。결론：pEE14.1-luc표체질립구건성공，위연구DNA역묘체내표체궤제화체내전천공체송조건우화전정료기출。
Objective: In order to study the delivery of DNA vaccine plasmid for over 10 kb by electroporation in vivo, we constructed the recombinant pEE14.1-luc, which can express the luciferase report gene. Methods: The luciferase gene was cut from pGL3-CMV vector, and inserted into pEE14.1-luc vector after filling in and purifica?tion. In vitro expression of the luciferase gene was identified by transient transfection to 293T cells, Western blot, flow cytometry and immunofluorescence. And in vivo expression of this gene was identified by in vivo imaging tech?nology. Results: The results indicated that pEE14.1-luc was constructed as we expected after the identification of microbial PCR and sequencing. The expression rate of luciferase was 22.41% tested by flow cytometry, and we could detect the expression of green fluorescence by immunofluorescence and a protein of 62 kD by Western blot. With in vivo imaging technology, we could also detect the expression of luciferase gene in mice in vivo. Conclu?sion: We conclude that pEE14.1-luc was successfully constructed, which lays a foundation for further studying the mechanism of in vivo expression of DNA vaccine and optimizing the in vivo conditions of delivery by electropora?tion.
